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Comparing Transcriptomic Points of Departure to Apical Effect Concentrations For Larval Fathead Minnow Exposed to Chemicals with Four Different Modes Of Action
It is postulated that below a transcriptomic-based point of departure, adverse effects are unlikely to occur, thereby providing a chemical concentration to use in screening level hazard assessment. The present study extends previous work describing a high-throughput fathead minnow assay that can provide full transcriptomic data after exposure to a test chemical. One-day post-hatch fathead minnows were exposed to ten concentrations of three representatives of four chemical modes of action: organophosphates, ecdysone receptor agonists, plant photosystem II inhibitors, and estrogen receptor agonists for 24 h. Concentration response modeling was performed on whole body gene expression data from each exposure, using measured chemical concentrations when available. Transcriptomic points of departure in larval fathead minnow were lower than apical effect concentrations across fish species but not always lower than toxic effect concentrations in other aquatic taxa like crustaceans and insects. The point of departure was highly dependent on measured chemical concentration which were often lower than the nominal concentration. Differentially expressed genes between chemicals within modes of action were compared and often showed statistically significant overlap. In addition, reproducibility between identical exposures using a positive control chemical (CuSO 4 ) and variability associated with the transcriptomic point of departure using in silico sampling were considered. Results extend a transcriptomic-compatible fathead minnow high-throughput assay for possible use in ecological hazard screening.
Short-Term Transcriptomic Points of Departure Are Consistent with Chronic Points of Departure for Three Organophosphate Pesticides across Mouse and Fathead Minnow
New approach methods (NAMs) can reduce the need for chronic animal studies. Here, we apply benchmark dose (concentration) (BMD(C))–response modeling to transcriptomic changes in the liver of mice and in fathead minnow larvae after short-term exposures (7 days and 1 day, respectively) to several dose/concentrations of three organophosphate pesticides (OPPs): fenthion, methidathion, and parathion. The mouse liver transcriptional points of departure (TPODs) for fenthion, methidathion, and parathion were 0.009, 0.093, and 0.046 mg/Kg-bw/day, while the fathead minnow larva TPODs were 0.007, 0.115, and 0.046 mg/L, respectively. The TPODs were consistent across both species and reflected the relative potencies from traditional chronic toxicity studies with fenthion identified as the most potent. Moreover, the mouse liver TPODs were more sensitive than or within a 10-fold difference from the chronic apical points of departure (APODs) for mammals, while the fathead minnow larva TPODs were within an 18-fold difference from the chronic APODs for fish species. Short-term exposure to OPPs significantly impacted acetylcholinesterase mRNA abundance (FDR p-value <0.05, |fold change| ≥2) and canonical pathways (IPA, p-value <0.05) associated with organism death and neurological/immune dysfunctions, indicating the conservation of key events related to OPP toxicity. Together, these results build confidence in using short-term, molecular-based assays for the characterization of chemical toxicity and risk, thereby reducing reliance on chronic animal studies.
Evaluating the Effects of Fly Ash Exposure on Fish Early Life Stages: Fathead Minnow Embryo-Larval Tests
On December 22, 2008, a dike containing fly ash and bottom ash in an 84-acre complex of the Tennessee Valley Authority's (TVA) Kingston Steam Plant in East Tennessee failed and released a large quantity of ash into the adjacent Emory River. Ash deposits extended as far as 4 miles upstream (Emory River mile 6) of the Plant, and some ash was carried as far downstream as Tennessee River mile 564 ({approx}4 miles downstream of the Tennessee River confluence with the Clinch River). A byproduct of coal burning power plants, fly ash contains a variety of metals and other elements which, at sufficient concentrations and in specific forms, can be toxic to biological systems. The effects of fly ash contamination on exposed fish populations depend on the magnitude and duration of exposure, with the most significant risk considered to be the effects of specific ash constituents, especially selenium, on fish early life stages. Uptake by adult female fish of fly ash constituents through the food chain and subsequent maternal transfer of contaminants to the developing eggs is thought to be the primary route of selenium exposure to larval fish (Woock and others 1987, Coyle and others 1993, Lemly 1999, Moscatello and others 2006), but direct contact of the fertilized eggs and developing embryos to ash constituents in river water and sediments is also a potential risk factor (Woock and others 1987, Coyle and others 1993, Jezierska and others 2009). To address the risk of fly ash from the Kingston spill to the reproductive health of downstream fish populations, ORNL has undertaken a series of studies in collaboration with TVA including: (1) a field study of the bioaccumulation of fly ash constituents in fish ovaries and the reproductive condition of sentinel fish species in reaches of the Emory and Clinch Rivers affected by the fly ash spill; (2) laboratory tests of the potential toxicity of fly ash from the spill area on fish embryonic and larval development (reported in the current technical manuscript); (3) additional laboratory experimentation focused on the potential effects of long-term exposures to fly ash on fish survival and reproductive competence; and (4) a combined field and laboratory study examining the in vitro developmental success of embryos and larvae obtained from fish exposed in vivo for over two years to fly ash in the Emory and Clinch Rivers. These fish reproduction and early life-stage studies are being conducted in conjunction with a broader biological monitoring program administered by TVA that includes a field study of the condition of larval fish in the Emory and Clinch Rivers along with assessments of water quality, sediment composition, ecotoxicological studies, terrestrial wildlife studies, and human and ecological risk assessment. Information and data generated from these studies will provide direct input into risk assessment efforts and will also complement and help support other phases of the overall biomonitoring program. Fish eggs, in general, are known to be capable of concentrating heavy metals and other environmental contaminants from water-borne exposures during embryonic development (Jezierska and others 2009), and fathead minnow embryos in particular have been shown to concentrate methylmercury (Devlin 2006) as well as other chemical toxicants. This technical report focuses on the responses of fathead minnow embryos to simple contact exposures to fly ash in laboratory toxicity tests adapted from a standard fathead minnow (Pimephales promelas) 7-d embryo-larval survival and teratogenicity test (method 1001.0 in EPA 2002) with mortality, hatching success, and the incidences of developmental abnormalities as measured endpoints.
Effects of Sediment Containing Coal Ash from the Kingston Ash Release on Embryo-Larval Development in the Fathead Minnow, Pimephales promelas (Rafinesque, 1820)
The largest environmental release of coal ash in U.S. history occurred in December 2008 with the failure of a retention structure at the Tennessee Valley Authority (TVA) Kingston Fossil Plant in East Tennessee. A byproduct of coal-burning power plants, coal ash is enriched in metals and metalloids such as selenium and arsenic with known toxicity to fish including embryonic and larval stages. The effects of contact exposure to sediments containing up to 78 % coal ash from the Kingston spill on the early development of fish embryos and larvae were examined in 7-day laboratory tests with the fathead minnow (Pimephales promelas). No significant effects were observed on hatching success, incidences of gross developmental abnormalities, or embryo-larval survival. Results suggest that direct exposures to sediment containing residual coal ash from the Kingston ash release may not present significant risks to fish eggs and larvae in waterways affected by the spill.
Reverse Engineering Adverse Outcome Pathways
The toxicological effects of many stressors are mediated through unknown, or poorly characterized, mechanisms of action. We describe the application of reverse engineering complex interaction networks from high dimensional omics data (gene, protein, metabolic, signaling) to characterize adverse outcome pathways (AOPs) for chemicals that disrupt the hypothalamus-pituitary-gonadal endocrine axis in fathead minnows. Gene expression changes in fathead minnow ovaries in response to 7 different chemicals, over different times, doses, and in vivo versus in vitro conditions were captured in a large data set of 868 arrays. We examined potential AOPs of the antiandrogen flutamide using two mutual information theory methods, ARACNE and CLR to infer gene regulatory networks and potential adverse outcome pathways. Representative networks from these studies were used to predict a network path from stressor to adverse outcome as a candidate AOP. The relationship of individual chemicals to an adverse outcome can be determined by following perturbations through the network in response to chemical treatment leading to the nodes associated with the adverse outcome. Identification of candidate pathways allows for formation of testable hypotheses about key biologic processes, biomarkers or alternative endpoints, which could be used to monitor an adverse outcome pathway. Finally, we identify the unique challenges facing the application of this approach in ecotoxicology, and attempt to provide a road map for the utilization of these tools. Key Words: mechanism of action, toxicology, microarray, network inference
Laboratory Studies of the Short-term Responses of Freshwater Fish to Electromagnetic Fields
Hydrokinetic energy technologies are being proposed as an environmentally preferred means of generating electricity from river and tidal currents. Among the potential issues that must be investigated in order to resolve environmental concerns are the effects on aquatic organisms of electromagnetic fields created by underwater generators and transmission cables. The behavioral responses of common freshwater fishes to static and variable electromagnetic fields (EMF) that may be emitted by hydrokinetic projects were evaluated in laboratory experiments. Various fish species were exposed to either static (DC) EMF fields created by a permanent bar magnet or variable (AC) EMF fields created by a switched electromagnet for 48 h, fish locations were recorded with a digital imaging system, and changes in activity level and distribution relative to the magnet position were quantified at 5-min intervals. Experiments with fathead minnows, redear sunfish, striped bass, lake sturgeon, and channel catfish produced mixed results. Except for fathead minnows there was no effect on activity level. Only redear sunfish and channel catfish exhibited a change in distribution relative to the position of the magnet with an apparent attraction to the EMF source. In separate experiments, rapid behavioral responses of paddlefish and lake sturgeon to onset of the AC field were recorded with high-speed video. Paddlefish did not react to a variable, 60-Hz magnetic field like that which would be emitted by an AC generator or cable, but lake sturgeon consistently responded to the variable, AC-generated magnetic field with a variety of altered swimming behaviors. These results will be useful for determining under what circumstances cables or generators need to be positioned to minimize interactions with sensitive species.
ROLE OF TOXICITY ASSESSMENT AND MONITORING IN MANAGING THE RECOVERY OF A WASTEWATER RECEIVING STREAM
We evaluate the roles of a long-term comprehensive toxicity assessment and monitoring program in management and for ecological recovery of a freshwater receiving stream impacted by industrial discharges and legacy contamination. National Pollution Discharge Elimination Permit (NPDES)-driven whole effluent toxicity (WET) tests using Ceriodaphnia and fathead minnows were conducted for more than twenty years to characterize wastewaters at the US National Nuclear Security Agency s Y-12 National Security Complex in Oak Ridge, Tennessee. Ambient toxicity tests also were conducted to assess water samples from EFPC, the stream receiving the wastewater discharges. The ambient tests were conducted as part of an extensive biological monitoring program that included routine surveys of fish, invertebrate and periphyton communities. WET testing, associated toxicant identification evaluations (TIEs), and ambient toxicity monitoring were instrumental in identifying toxicants and their sources at the Y-12 Complex, guiding modifications to wastewater treatment procedures, and assessing the success of various pollution-abatement actions. Through time, as requirements changed and water quality improved, the toxicity monitoring program became more focused. Ambient testing with Ceriodaphnia and fathead minnow larvae also was supplemented with less-standardized but more-sensitive alternative laboratory and in situ bioassays. The Y-12 Complex biological monitoring experience demonstrates the significant roles effluent and ambient toxicity testing can have in controlling and managing toxic discharges to receiving waters. It also emphasizes the value of supplementing WET and standardized ambient toxicity tests with alternative laboratory and in situ toxicity tests tailored to address specific problems.
Laboratory Studies of the Effects of Static and Variable Magnetic Fields on Freshwater Fish
There is considerable interest in the development of marine and hydrokinetic energy projects in rivers, estuaries, and coastal ocean waters of the United States. Hydrokinetic (HK) technologies convert the energy of moving water in river or tidal currents into electricity, without the impacts of dams and impoundments associated with conventional hydropower or the extraction and combustion of fossil fuels. The Federal Energy Regulatory Commission (FERC) maintains a database that displays the geographical distribution of proposed HK projects in inland and tidal waters (FERC 2012). As of March 2012, 77 preliminary permits had been issued to private developers to study HK projects in inland waters, the development of which would total over 8,000 MW. Most of these projects are proposed for the lower Mississippi River. In addition, the issuance of another 27 preliminary permits for HK projects in inland waters, and 3 preliminary permits for HK tidal projects (totaling over 3,100 MW) were under consideration by FERC. Although numerous HK designs are under development (see DOE 2009 for a description of the technologies and their potential environmental effects), the most commonly proposed projects entail arrays of rotating devices, much like submerged wind turbines, that are positioned in the high-velocity (high energy) river channels. The many diverse HK designs imply a diversity of environmental impacts, but a potential impact common to most is the effect on aquatic organisms of electromagnetic fields (EMF) created by the projects. The submerged electrical generator will emit an EMF into the surrounding water, as will underwater cables used to transmit electricity from the generator to the shore, between individual units in an array (inter-turbine cables), and between the array and a submerged step-up transformer. The electric current moving through these cables will induce magnetic fields in the immediate vicinity, which may affect the behavior or viability of fish and benthic invertebrates (Gill et al. 2005, 2009). It is known that numerous marine and freshwater organisms are sensitive to electrical and magnetic fields, often depending on them for such diverse activities as prey location and navigation (DOE 2009; Normandeau et al. 2011). Despite the wide range of aquatic organisms that are sensitive to EMF and the increasing numbers of underwater electrical transmitting cables being installed in rivers and coastal waters, little information is available to assess whether animals will be attracted, repelled, or unaffected by these new sources of EMF. This knowledge gap is especially significant for freshwater systems, where electrosensitive organisms such as paddlefish and sturgeon may interact with electrical transmission cables. We carried out a series of laboratory experiments to test the sensitivity of freshwater fish and invertebrates to the levels of EMF that are expected to be produced by HK projects in rivers. In this context, EM fields are likely to be emitted primarily by generators in the water column and by transmission cables on or buried in the substrate. The HK units will be located in areas of high-velocity waters that are used as only temporary habitats for most riverine species, so long-term exposure of fish and benthic invertebrates to EMF is unlikely. Rather, most aquatic organisms will be briefly exposed to the fields as they drift downstream or migrate upstream. Because the exposure of most aquatic organisms to EMF in a river would be relatively brief and non-lethal, we focused our investigations on detecting behavioral effects. For example, attraction to the EM fields could result in prolonged exposures to the fields or the HK rotor. On the other hand, avoidance reactions might hinder upstream migrations of fish. The experiments reported here are a continuation of studies begun in FY 2010, which focused on the potential effects of static magnetic fields on snails, clams, and fathead minnows (Cada et al. 2011). Those experiments found little indication that the behaviors of these freshwater species were altered by the static magnetic fields that would be created by submerged, direct current (DC)-transmitting electrical cables expected to be used by the HK developers. Laboratory experiments in FY 2011 examined the responses of additional fish species (sunfish, striped bass, and channel catfish) to the static magnetic fields. In addition, the effects of variable magnetic fields (that would be created by the HK generators and AC-transmitting cables) on swimming behavior of two electrosensitive fish species (paddlefish and lake sturgeon) were studied.
Maternal exposure to carbamazepine at environmental concentrations can cross intestinal and placental barriers
Psychoactive pharmaceuticals have been found as teratogens at clinical dosage during pregnancy. These pharmaceuticals have also been detected in minute (ppb) concentrations in drinking water in the US, and are environmental contaminants that may be complicit in triggering neurological disorders in genetically susceptible individuals. Previous studies have determined that psychoactive pharmaceuticals (fluoxetine, venlafaxine and carbamazepine) at environmentally relevant concentrations enriched sets of genes regulating development and function of the nervous system in fathead minnows. Altered gene sets were also associated with potential neurological disorders, including autism spectrum disorders (ASD). Subsequent in vitro studies indicated that psychoactive pharmaceuticals altered ASD-associated synaptic protein expression and gene expression in human neuronal cells. However, it is unknown if environmentally relevant concentrations of these pharmaceuticals are able to cross biological barriers from mother to fetus, thus potentially posing risks to nervous system development. The main objective of this study was to test whether psychoactive pharmaceuticals (fluoxetine, venlafaxine, and carbamazepine) administered through the drinking water at environmental concentrations to pregnant mice could reach the brain of the developing embryo by crossing intestinal and placental barriers. We addressed this question by adding {sup 2}H-isotope labeled pharmaceuticals to the drinking water of female mice for 20 days (10 pre-and 10 post–conception days), and quantifying {sup 2}H-isotope enrichment signals in the dam liver and brain of developing embryos using isotope ratio mass spectrometry. Significant levels of {sup 2}H enrichment was detected in the brain of embryos and livers of carbamazepine-treated mice but not in those of control dams, or for fluoxetine or venlafaxine application. These results provide the first evidence that carbamazepine in drinking water and at typical environmental concentrations is transmitted from mother to embryo. Our results, combined with previous evidence that carbamazepine may be associated with ASD in infants, warrant the closer examination of psychoactive pharmaceuticals in drinking water and their potential association with neurodevelopmental disorders.
Fish Toxicity Non-Hazardous Waste GAC Data
Attached are the test data generated from the analysis of the Fathead Minnow Hazardous Waste Screen bioassay sample. All testing was conducted under the direct supervision of Joseph A. LeMay.